Journal: Scientific Reports

Article Title: The latent dedifferentiation capacity of newt limb muscles is unleashed by a combination of metamorphosis and body growth

doi: 10.1038/s41598-022-15879-z

Figure Lengend Snippet: Muscle fiber nucleus-tracking system. ( a ) A transgene cassette for conditional gene expression in muscle fibers. To label the nuclei of skeletal muscle fibers and mono-SMFCs, a nuclear-localized derivative of mCherry (N-mCherry) was used. This cassette expresses inducible Cre-recombinase (CreER T2 ) in mature skeletal muscle fibers under the control of the cardiac actin promoter ( CarA ). In the presence of 4-hydroxy tamoxifen (4-OHT), a fluorescent marker, which is expressed under the control of the universal promoter CAGGs , is switched from EGFP to N-mCherry via the loxP system. CAGGs makes it possible to monitor skeletal muscle fibers and their derivatives during limb regeneration. I-SceI I- Sce I meganuclease recognition sequence, 2xHS4 insulator sequence. ( b ) Experiment timeline. The construct was injected into one-cell stage embryos of albino F1 and wild-type F0. To induce recombination, swimming larvae at St. 53 (1–2 months old), which only had three of five digits on the hind limbs, were incubated in tap water containing 4-OHT for 24 h. ( c – f ) Representative images showing the expression of EGFP and N-mCherry in the forelimbs of albino juveniles. A large number of N-mCherry nuclei were observed along the muscle fibers. ( g ) Labeling specificity. A representative confocal image of transverse sections of limb muscle showing uniform expression of EGFP. Immunohistochemistry showed that satellite cells, which are resident myogenic stem cells, that express Pax7 (blue), were never labelled by N-mCherry. ( h ) Albino F1 newts grown in the laboratory. Scale bar: 1 mm ( c – e ); 200 μm ( f ); 100 μm ( g ); 3 cm ( h ).

Article Snippet: This cassette comprises the Cre driver and LoxP reporter constructs in opposite directions (Fig. a): the reporter construct CAGGs > [EGFP]N-mCherry expresses EGFP, whose gene is flanked by loxP sites and followed by the N-mCherry gene, under the control of a universal promoter ( CAGGs ); the driver construct CreER T2 < CarA expresses inducible Cre recombinase (Cre-ER T2 ) under the control of a cardiac actin promoter ( CarA ; from M11 cardiac actin promoter pCarA, Addgene 17148 ), allowing the specific expression of N-mCherry in mature skeletal muscle fibers of limbs via Cre-mediated recombination of the floxed EGFP in the presence of an inducer tamoxifen.

Techniques: Gene Expression, Control, Marker, Sequencing, Construct, Injection, Incubation, Expressing, Labeling, Immunohistochemistry

Journal: Biomolecules

Article Title: Thoracic Aortic Aneurysm Development Is Dependent on Membrane Type-1 Matrix Metalloproteinase Activity and Abundance

doi: 10.3390/biom16020237

Figure Lengend Snippet: Changes in aortic diameter and MT1-MMP abundance over time after TAA induction in MT1-MMP fl/fl (control) and Col1A2-Cre(ERT2) + × MT1-MMP fl/fl (FbMT1KO) mice. Mice from both groups received TAA surgery. At 4 weeks post-TAA, mice were treated daily for 5 days with tamoxifen (75 mg/kg/day ip, corn oil). Aortic diameter was determined at baseline and terminal surgery in each animal using digital micrometry. ( A ) Representative images of aortas at baseline and 16 weeks post-TAA. ( B ) Aortic diameter values were expressed as percent change from baseline (100%) in each animal. Individual data points are shown on the graph (C57BL/6 floxed-MT1-MMP (control): light purple symbols; FbMT1KO: dark purple symbols). Aortic diameters were increased over baseline values at the 4-, 8-, and 16-wk time points for both the wild-type and FbMT1KO mice (# p < 0.001 at each time point for both strains). Aortic diameters at the 16-week time point in FbMT1KO mice were significantly different from control mice (* p < 0.001). ( C ) Aortic tissue homogenates combined from two aortas from indicated mice at indicated times were subjected to immunoblotting using antibodies for MT1-MMP and β-actin to determine respective protein levels. Images are representative from three independent experiments. Results reflect a minimum of 6 animals/experimental group in each strain. Original figures can be found in .

Article Snippet: The fibroblast-specific, tamoxifen-inducible, Cre-recombinase mice (Col1A2-Cre(ERT2)) were constructed by inserting a 6.4 kb fragment of the murine pro-alpha2(I) collagen ( Col1A2 ) gene (containing the promoter/enhancer region described by Bou-Gharios et al. [ ] and Denton et al. [ ], which strongly directs transgene expression in fibroblasts) upstream of human β-globin intron 2, the Cre-ERT2 ORF, and an SV40-polyA sequence into a pGL3 vector (Promega, Madison, WI, USA) (Taconic-Artemis, Germantown, NY, USA) ( ).

Techniques: Control, Western Blot

Journal: bioRxiv

Article Title: A therapeutically targetable NOTCH1-SIRT1-KAT7 axis in T-cell Leukemia

doi: 10.1101/2022.05.21.492944

Figure Lengend Snippet: SIRT1 is overexpressed in T-ALL downstream of a NOTCH1-bound enhancer. A, Box-plot showing SIRT1 expression among T-ALL samples (n=57) and physiological thymocyte subsets (n=21) . Quantile normalization was performed across samples. Boxes represent first and third quartiles and the line represents the median. Whiskers represent the upper and lower limits ( P <0.001 using Mann-Whitney U-Test; FDR<0.05 using Benjamini-Hochberg correction). B, Western blot analysis of SIRT1 and ACTIN expression in human peripheral blood mononuclear cells (PBMNC), CD4+ T-cells or normal human thymocytes, as compared to human T-ALL cell lines. C, GSI washout experiments in CUTLL1 T-ALL cells, treated with GSI (Compound E, 1μM) for 3 days, washed twice, and incubated 4h in the presence or absence of 20μM cycloheximide . To control for GSI “off-NOTCH” effects, cells were also transduced with a dominant-negative MAML1 (DN-MAML1). (n=3 per condition; *** P < 0.005 using two-tailed Student t -test; NS, not significant). D, Western blot analysis of NOTCH1 (ICN1), SIRT1 and ACTIN expression in triplicates from DND41 or HPB-ALL human T-ALL cells treated with DBZ (250nM) for 3 days or mouse T-ALL cells treated with DBZ (250nM) for 24h. E, Epigenetic profiling around the SIRT1 promoter in human T-ALL showing ChIP-seq tracks in human T-ALL cell lines and ATAC-seq tracks in human T-ALL primary samples. N-Se enhancer highlighted in orange. F, Luciferase reporter activity in JURKAT cells of a pGL4 promoter empty construct (pGL4-Luc), a pGL4 promoter plus the human N-Se enhancer in the forward (NSe(+)-Luc) or reverse (NSe(-)-Luc) orientation. Data from three independent electroporation replicates are shown. *** P < 0.005 using two-tailed Student t -test. G, Genotyping of JURKAT single-cell clones harboring a N-Se homozygous deletion. JURKAT cells not electroporated (WT) are shown as controls. H, SIRT1 protein expression levels via western blot analysis in JURKAT control cells or four independent JURKAT single-cell clones with N-Se homozygous deletion. I, SIRT1 protein expression levels via western blot analysis in DND41 cells harboring either a dCas9-VP64 or dCas9-KRAB construct, and infected with gRNAs targeting either the SIRT1 promoter transcriptional start site (TSS) or two independent gRNAs targeting N-Se.

Article Snippet: For leukemia-progression studies, already generated E-NOTCH1-GFP-induced or HD P-NOTCH1-GFP-induced Sirt1 flox/flox -Rosa26 Cre-ERT2/+ leukemias (1 x 10 6 leukemia cells) were transplanted from primary recipients into sub-lethally irradiated (4.5 Gy) 6-8- week-old secondary recipient C57BL/6 mice (Taconic Farms) by retro-orbital injection.

Techniques: Expressing, MANN-WHITNEY, Western Blot, Incubation, Transduction, Dominant Negative Mutation, Two Tailed Test, ChIP-sequencing, Luciferase, Activity Assay, Construct, Electroporation, Clone Assay, Infection

Journal: bioRxiv

Article Title: A therapeutically targetable NOTCH1-SIRT1-KAT7 axis in T-cell Leukemia

doi: 10.1101/2022.05.21.492944

Figure Lengend Snippet: SIRT1 inhibition shows antileukemic and synergistic effects with NOTCH1 inhibition. A-B, SIRT1 mRNA expression levels ( A ) and protein expression levels ( B ) in DND41 cells harboring two independent doxycycline-inducible shRNAs targeting SIRT1 with concomitant GFP expression or a non-targeting shRNA control, 3 days after doxycycline induction. C, Proliferation curve (left) and cell quantification at day 9 (right) of DND41 cells upon Doxycycline-induced expression of a control shRNA or shRNAs targeting SIRT1 . D-E , Representative flow cytometry plots from triplicate samples of annexin V (apoptotic cells) and 7-AAD (dead cells) staining ( D ) and quantification of apoptosis ( E ) of DND41 cells 9 days after Doxycycline-induced expression of a control shRNA or shRNAs targeting SIRT1 . Numbers in quadrants indicate percentage of cells. F, Proliferation curve (left) and cell quantification at day 9 (right) of DND41 cells treated with vehicle (DMSO), EX-527 (90μM), DBZ (250nM), or EX-527 and DBZ in combination. G , Isobologram analysis of DBZ and EX-527 treatment after 6 days in DND41 cells. The value for the combination index at ED50 is marked in blue. The ED50 for each drug is marked in black. H-I , Representative flow cytometry plots from triplicate samples of annexin V (apoptotic cells) and 7-AAD (dead cells) staining ( H ) and quantification of apoptosis ( I ) of DND41 cells treated with vehicle (DMSO), EX-527(90μM), DBZ (250nM) or EX-527 and DBZ in combination at Day 9. Numbers in quadrants indicate percentage of cells. * P < 0.05 and *** P < 0.005 in Figs. , and 1F using one-way analysis of variance (ANOVA). * P < 0.05 ** P < 0.01 and *** P < 0.005 in Figs. and using two-way ANOVA for multiple comparisons.

Article Snippet: For leukemia-progression studies, already generated E-NOTCH1-GFP-induced or HD P-NOTCH1-GFP-induced Sirt1 flox/flox -Rosa26 Cre-ERT2/+ leukemias (1 x 10 6 leukemia cells) were transplanted from primary recipients into sub-lethally irradiated (4.5 Gy) 6-8- week-old secondary recipient C57BL/6 mice (Taconic Farms) by retro-orbital injection.

Techniques: Inhibition, Expressing, shRNA, Flow Cytometry, Staining

Journal: bioRxiv

Article Title: A therapeutically targetable NOTCH1-SIRT1-KAT7 axis in T-cell Leukemia

doi: 10.1101/2022.05.21.492944

Figure Lengend Snippet: SIRT1 promotes T-ALL development and confers resistance to NOTCH1 inhibition in vivo . A, Schematic of retroviral-transduction protocol for the generation of NOTCH1-induced T-ALLs from Sirt1 -overexpressing ( Sirt1 TG ) or wild-type control littermate ( Sirt1 WT ) mice. B, Kaplan-Meier curves of mice transplanted with E-NOTCH1 infected Sirt1 WT and Sirt1 TG hematopoietic progenitors (n=10 per genotype). * P < 0.05 value was calculated using the log-rank test. C, Western blot analysis of SIRT1 and ACTIN expression in leukemic spleens from terminally ill mice from survival curve in B. D, Schematic of retroviral-transduction protocol for the generation of NOTCH1-induced T-ALLs from inducible Sirt1 -conditional knockout mice. Two days upon transplantation of NOTCH1-infected Sirt1 flox/flox -Rosa26 Cre-ERT2/+ progenitors, mice were treated with corn oil vehicle ( Sirt1 +/+ ) or tamoxifen ( Sirt1 -/- ), in order to induce isogenic loss of Sirt1 . E, Kaplan-Meier curves of mice transplanted with NOTCH1-infected Sirt1 flox/flox -Rosa26 Cre-ERT2/+ progenitors (n=10 and treated with vehicle ( Sirt1 +/+ ) or tamoxifen ( Sirt1 -/- ) as in D (n=10 per genotype). * P < 0.05 value was calculated using the log-rank test. F, Western blot analysis of SIRT1 and ACTIN expression in leukemic spleens from terminally ill mice from survival curve in E. G, Schematic for transduction of either wild-type Sirt1 or a deacetylase-dead H355A Sirt1 mutant concomitantly expressing the mCherry fluorescent protein in NOTCH1-induced GFP+ primary T-ALL cells followed by transplantation into mice, which were subsequently treated daily with DBZ in vivo . H, Peripheral blood leukemia infiltration in mice harboring NOTCH1-induced T-ALL cells expressing mCherry and Sirt1 wild-type or H355A-mutant upon continuous daily treatment with DBZ. Changes in leukemia cell counts of noninfected (mCherry-negative) cells are shown as an internal control. Error bars, median ± s.d.; P values were calculated using two-tailed Student t -test (n=5 mice per group); NS, not significant. I, C, Western blot analysis of SIRT1 and GAPDH expression in leukemic spleens from non-DBZ-treated, terminally ill mice from G.

Article Snippet: For leukemia-progression studies, already generated E-NOTCH1-GFP-induced or HD P-NOTCH1-GFP-induced Sirt1 flox/flox -Rosa26 Cre-ERT2/+ leukemias (1 x 10 6 leukemia cells) were transplanted from primary recipients into sub-lethally irradiated (4.5 Gy) 6-8- week-old secondary recipient C57BL/6 mice (Taconic Farms) by retro-orbital injection.

Techniques: Inhibition, In Vivo, Transduction, Infection, Western Blot, Expressing, Knock-Out, Transplantation Assay, Histone Deacetylase Assay, Mutagenesis, Two Tailed Test

Journal: bioRxiv

Article Title: A therapeutically targetable NOTCH1-SIRT1-KAT7 axis in T-cell Leukemia

doi: 10.1101/2022.05.21.492944

Figure Lengend Snippet: Secondary loss of SIRT1 in established leukemias leads to antileukemic and synergistic effects with NOTCH1 inhibition in vivo . A, Schematic of retroviral-transduction protocol for the generation of NOTCH1-induced T-ALLs from inducible Sirt1 -conditional knockout mice, followed by transplant into secondary recipients treated with vehicle ( Sirt1 +/+ ) or tamoxifen ( Sirt1 -/- ) and vehicle or DBZ. B, Kaplan-Meier survival curves of mice harboring Sirt1 -positive and Sirt1 -deleted isogenic leukemias treated with 4 cycles of vehicle or DBZ (5 mg/kg) on a 4-days-ON (red blocks) and 3-days-OFF schedule (log-rank test; ** P < 0.01; *** P < 0.005; n = 10 per group). C, Western blot analysis of SIRT1 and ACTIN expression in leukemic spleens from terminally ill mice from survival curve in B. D-E , Quantitative RT-PCR analysis of Sirt1 mRNA expression (D) and western blot analysis of SIRT1 protein levels (E) in tumor cells isolated from E-NOTCH1-induced Sirt1 conditional knockout leukemia–bearing mice 48 h after being treated with vehicle only ( Sirt1 +/+ ) or tamoxifen ( Sirt1 -/- ) in vivo . F-G, Tumor burden in E-NOTCH1-induced Sirt1 conditional knockout leukemia–bearing mice 48 h after being treated with vehicle only ( Sirt1 +/+ ) or tamoxifen ( Sirt1 -/- ) in vivo as revealed by total spleen weight (F) and total spleen cell numbers (G). H-I , Representative flow cytometry plots from of annexin V (apoptotic cells) and 7-AAD (dead cells) staining ( H ) and quantification of apoptosis ( I ) in leukemic spleens from E-NOTCH1-induced Sirt1 conditional knockout leukemia– bearing mice 48 h after being treated with vehicle only ( Sirt1 +/+ ) or tamoxifen ( Sirt1 -/- ) in vivo . (n = 5 per treatment; ** P < 0.01 and *** P < 0.005 in using two-tailed Student t -test).

Article Snippet: For leukemia-progression studies, already generated E-NOTCH1-GFP-induced or HD P-NOTCH1-GFP-induced Sirt1 flox/flox -Rosa26 Cre-ERT2/+ leukemias (1 x 10 6 leukemia cells) were transplanted from primary recipients into sub-lethally irradiated (4.5 Gy) 6-8- week-old secondary recipient C57BL/6 mice (Taconic Farms) by retro-orbital injection.

Techniques: Inhibition, In Vivo, Transduction, Knock-Out, Western Blot, Expressing, Quantitative RT-PCR, Isolation, Flow Cytometry, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: A therapeutically targetable NOTCH1-SIRT1-KAT7 axis in T-cell Leukemia

doi: 10.1101/2022.05.21.492944

Figure Lengend Snippet: Metabolic consequences of secondary loss of SIRT1 in established leukemias in vivo . A, Western blot analysis of AMPK protein levels and activation, 4E-BP1 levels and activation, and ATF4 levels in tumor cells isolated from E-NOTCH1-induced Sirt1 conditional knockout leukemia–bearing mice 48 h after being treated with vehicle only ( Sirt1 +/+ ) or tamoxifen ( Sirt1 -/- ) in vivo . B, Significantly altered metabolites (upregulated in red, downregulated in blue) upon tamoxifen-induced isogenic loss of Sirt1 in leukemic spleens from mice treated as in A, ranked by P value (–log10 transformed). C-D, Relative abundance of indicated glycolytic intermediates (C) or glutamine/aspartate-related metabolites (D) upon tamoxifen-induced isogenic loss of Sirt1 in leukemic spleens from mice treated as in A. (n = 5 per treatment; * P < 0.05, ** P < 0.01 and *** P < 0.005 in Figs. 5A-D using two-tailed Student t -test). E, Oxygen consumption rate (OCR) in response to the indicated mitochondrial inhibitors in a E-NOTCH1-induced Sirt1 conditional knockout leukemia-derived cell line under basal conditions or 2-days after 4-Hydroxytamoxifen-induced isogenic loss of Sirt1 , measured in real time using a Seahorse XF24 instrument. Data are presented as +/- SD of n = 5 wells.

Article Snippet: For leukemia-progression studies, already generated E-NOTCH1-GFP-induced or HD P-NOTCH1-GFP-induced Sirt1 flox/flox -Rosa26 Cre-ERT2/+ leukemias (1 x 10 6 leukemia cells) were transplanted from primary recipients into sub-lethally irradiated (4.5 Gy) 6-8- week-old secondary recipient C57BL/6 mice (Taconic Farms) by retro-orbital injection.

Techniques: In Vivo, Western Blot, Activation Assay, Isolation, Knock-Out, Transformation Assay, Two Tailed Test, Derivative Assay

Journal: bioRxiv

Article Title: A therapeutically targetable NOTCH1-SIRT1-KAT7 axis in T-cell Leukemia

doi: 10.1101/2022.05.21.492944

Figure Lengend Snippet: Secondary loss of SIRT1 leads to hyperacetylation of KAT7 and a transcriptional signature driven by KAT7 inhibition. A, Schematic representation of acetyl-proteomic experiments in tumor cells isolated from E-NOTCH1 or HD P-NOTCH1- induced Sirt1 conditional knockout leukemia–bearing mice 48 h after being treated with vehicle only ( Sirt1 +/+ ) or tamoxifen ( Sirt1 -/- ) in vivo . Venn diagram shows significantly hyperacetylated targets consistently found upon SIRT1 loss in both leukemias. B, Heatmap representation of the top differentially expressed genes between control ( Sirt1 +/+ ) and tamoxifen-treated ( Sirt1 -/- ) Sirt1 conditional knockout E-NOTCH1–induced leukemias. Cutoffs used: Wald statistic < −8 or > 8; P-adjusted value < 0.005; sorted based on mean expression levels. Scale bar shows color-coded differential expression, with red indicating higher levels of expression and blue indicating lower levels of expression. C, GSEA of genes regulated by KAT7 in vehicle only–treated ( Sirt1 +/+ ) compared with tamoxifen–treated ( Sirt1 -/- ) E-NOTCH1-induced Sirt1 conditional knockout leukemia cells in vivo . D, Western blot analysis (left) and quantification (right) of H4 total protein levels and H4K12ac levels in tumor cells isolated from E-NOTCH1- induced Sirt1 conditional knockout leukemia–bearing mice 2 days after being treated with vehicle only ( Sirt1 +/+ ) or tamoxifen ( Sirt1 -/- ) in vivo . ** P < 0.01 using two-tailed Student t -test. E, Western blot analysis (left) and quantification (right) of H4 total protein levels and H4K12ac levels in DND41 cells harboring two independent doxycycline-inducible shRNAs targeting SIRT1 with concomitant GFP expression or a non-targeting shRNA control, 3 days after doxycycline induction. F, Bar graph showing the number of significantly downregulated (blue) or upregulated (red) H4K12ac-containing genomic regions from H4K12ac ChIP-seq analyses in tumor cells isolated from E-NOTCH1-induced Sirt1 conditional knockout leukemia–bearing mice 48 h after being treated with vehicle only ( Sirt1 +/+ ) or tamoxifen ( Sirt1 -/- ) in vivo . G, Volcano plot showing H4K12ac downregulated region genes (shrunken LFC < -0.3) in blue (n=2319) and the rest of the regions in light grey. Horizontal dashed line corresponds to the adjusted P value threshold of 0.001. Vertical dashed lines correspond to log 2 FC changes of +0.25 and -0.25. 303/2319 were significantly downregulated at the gene expression level (hypergeometric test P value = 9.48E-40).

Article Snippet: For leukemia-progression studies, already generated E-NOTCH1-GFP-induced or HD P-NOTCH1-GFP-induced Sirt1 flox/flox -Rosa26 Cre-ERT2/+ leukemias (1 x 10 6 leukemia cells) were transplanted from primary recipients into sub-lethally irradiated (4.5 Gy) 6-8- week-old secondary recipient C57BL/6 mice (Taconic Farms) by retro-orbital injection.

Techniques: Inhibition, Isolation, Knock-Out, In Vivo, Expressing, Western Blot, Two Tailed Test, shRNA, ChIP-sequencing

Journal: bioRxiv

Article Title: A therapeutically targetable NOTCH1-SIRT1-KAT7 axis in T-cell Leukemia

doi: 10.1101/2022.05.21.492944

Figure Lengend Snippet: KAT7 partially mediates the antileukemic effects of loss of SIRT1. A, Relative cell proliferation of a E-NOTCH1-induced Sirt1 conditional knockout leukemia-derived cell line upon treatment with ethanol ( Sirt1 +/+ ) or 4-Hydroxytamoxifen ( Sirt1 -/- ) and different concentrations of the KAT7 inhibitor WM-3835 in vitro . * P < 0.05 and *** P < 0.005 using two-tailed Student t -test. B, Western blot analysis (left) and quantification (right) of a E-NOTCH1-induced Sirt1 conditional knockout leukemia-derived cell line infected with an empty vector (control) or with constructs overexpressing wild-type KAT7, K277Q mutant KAT7 or K277R mutant KAT7. C, Quantification of total cell numbers 6 days after 4-hydroxytamoxifen-induced loss of SIRT1 in cells overexpressing an empty vector or different KAT7 versions from B. D, Quantification of apoptosis (Annexin V-positive cells) 6 days after 4-hydroxytamoxifen-induced loss of SIRT1 in cells overexpressing an empty vector or different KAT7 versions from B. * P < 0.05 and ** P < 0.01 in Figs. 7C-D using 2-way analysis of variance (ANOVA) for multiple comparisons.

Article Snippet: For leukemia-progression studies, already generated E-NOTCH1-GFP-induced or HD P-NOTCH1-GFP-induced Sirt1 flox/flox -Rosa26 Cre-ERT2/+ leukemias (1 x 10 6 leukemia cells) were transplanted from primary recipients into sub-lethally irradiated (4.5 Gy) 6-8- week-old secondary recipient C57BL/6 mice (Taconic Farms) by retro-orbital injection.

Techniques: Knock-Out, Derivative Assay, In Vitro, Two Tailed Test, Western Blot, Infection, Plasmid Preparation, Construct, Mutagenesis